ABSTRACT Nontuberculous mycobacteria (NTM) are ubiquitous organisms that cause chronic pulmonary infection, particularly in individuals with pre-existing lung disease, such as COPD and cystic fibrosis. With an increasing incidence, pulmonary NTM disease has far surpassed that of tuberculosis in the US. Mycobacterium avium (M. avium) is responsible for the vast majority of morbidity and mortality from pulmonary NTM disease, while M. abscessus is one of the most virulent and feared subspecies of NTM, given its high levels of drug- resistance and notoriously poor treatment outcomes despite prolonged antibiotic courses. While there are numerous known mutations that are associated with drug resistance in NTM, their predictive value remains vague. Sadly, antibiotic selection in NTM remains an art and not a science, which should not be the case in the current era of whole-genome sequencing and precision medicine. This application seeks to harness the advances of genomics and personalized medicine to improve the health of patients with NTM disease. My research to date has revealed new insights into the evolutionary patterns of resistance in M. tuberculosis, as well as novel molecular mechanisms of resistance to cycloserine and beta-lactams. My fellowship research has led to 10 publications, five of which as first-author, including two first-author papers in Nature Genetics, and one first-author paper in PLoS Medicine. My prior experience with mycobacteriology, genomics of drug resistance, and clinical training in pulmonary medicine combined with support from my eminent co-mentors, make me uniquely qualified to undertake these innovative new studies of mechanisms of resistance in clinically relevant NTM. In this proposal, I will establish a prospective observational cohort of patients with NTM at the Johns Hopkins NTM Clinic. In a longitudinal manner, I will monitor the clinical progress of cohort members as well as collect serial mycobacterial isolates at each clinic visit. Both retrospectively biobanked strains and prospectively collect clinical isolates of M. avium and M. abscessus will be characterized by extensive phenotypic drug susceptibility testing in addition to whole-genome sequencing. Pairing phenotypic and genomic data will enable me to identify genomic elements that highly correlate with resistance. With a focus on macrolides, rifampin, oxazolidonones and beta-lactams, I will perform molecular validation studies using gene replacement to validate that these candidate genetic features do in fact confer phenotypic resistance to their respective drug. In my final aim, I will seek to investigate the yet unknown molecular basis for rifampin resistance in M. avium and M. abscessus and the potential for efflux pump inhibition to restore susceptibility to this key drug. At the end of these five years, these studies have a high probability of leading to rapid PCR- based diagnostics and for drug resistance in these pathogens.